Keiko Momma

Molecular Identification of Oligoalginate Lyase of Sphingomonas sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.

Safety assessment of genetically engineered potatoes with designed soybean glycinin: compositional analyses of the potato tubers and digestibility of the newly expressed protein in transgenic potatoes

The compositions of transgenic potatoes with intact and modified genes for soybean glycinin were compared with those of control potatoes; non-transgenic or transgenic ones with a vector. The expression levels of glycinin proteins in the transgenic potato tubers were approximately 12–31 mg g−1-total soluble protein. With reference to six major components, moisture, protein, lipid, fiber, ash and carbohydrate, there were no significant differences between transgenic and control potatoes. The levels of glycoalkaloids in transgenic potato tubers, as well as in transgenic controls, increased in comparison with those in non-transgenic ones, though the level was within the permitted limit. The modified glycinin expressed in the transgenic potato tuber was digested under the simulated gastric conditions. From these results, the transgenic potatoes with intact and modified glycinin genes are considered to be as safety utilization for food as non-transgenic potatoes. © 1999 Society of Chemical Industry

A Novel Bacterial ATP-Binding Cassette Transporter System That Allows Uptake of Macromolecules

A gram-negative bacterium, Sphingomonas sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation. algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS(His6)) overexpressed in Escherichia coli and purified by Ni(2+) affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.

Sphingomonas sp. A1 lyase active on both poly-β-D-mannuronate and heteropolymeric regions in alginate

Alginate lyase III of Sphingomonas sp. A1 cleaved the glycosidic linkage of polymannuronate and heteropolymeric region composed of mannuronate and guluronate, but was inert on polyguluronate. The enzyme was observed to act endolytically, interact with tetrasaccharide in alginate, and form di- and trisaccharides as final products. This result suggests that the enzyme recognizes the unit of tetrasaccharide in alginate and cleaves the middle linkage of the tetrasaccharide.

Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 degrees C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, alpha and beta. A deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.

Direct interaction of Cucurbitacin E isolated from Alsomitra macrocarpa to actin filament

A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed.

Structure and function of bacterial super-biosystem responsible for import and depolymerization of macromolecules.

Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a “superchannel”, as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.

Enzymatic and genetic bases on assimilation, depolymerization, and transport of heteropolysaccharides in bacteria

When microorganisms utilize macromolecules for their growth, they commonly produce extracellular depolymerization enzymes and then incorporate the depolymerized low-molecular-weight products. Assimilation of heteropolysaccharides (gellan and xanthan) by Bacillus sp. GL1 depends on this generally accepted mechanism. On the other hand, Sphingomonas sp. A1 represents an unexplored specific and interesting system for macromolecule assimilation. In the presence of heteropolysaccharide (alginate), the bacterium forms a mouthlike pit on its cell surface and directly incorporates the macromolecule using a novel ATP-binding cassette transporter (ABC transporter). In this review, we discuss enzymatic and genetic bases on the depolymerization and assimilation routes of heteropolysaccharides in bacteria, with particular emphasis on the novel incorporation system for macromolecules, characteristic post-translational modification processes of polysaccharide lyases and on the mouthlike pit structure on the bacterial cell surface.

Special cell surface structure, and novel macromolecule transport/depolymerization system of Sphingomonas sp A1.

A bacterium isolated from soil as an alginate lyase producer shows characteristic morphological and taxonomical properties consistent with being classified in the genus Sphingomonas. The bacterium utilizes high molecular weight (HMW)-alginate for growth by depolymerization of the polymer with intracellular alginate lyases, which are generated from a common precursor protein through autoregulated post-translational modifications. Electron microscopic observations of the cell surface and of thin sections of cells grown on HMW-alginate revealed dynamic changes in both cell surface and membrane structures. The most remarkable change is recognized in the formation of mouth-like pits which open and close depending on the presence or absence of HMW-alginate. Enzymatic and genetic analyses of HMW-alginate incorporation processes confirmed the presence of a pit-dependent and macromolecule-specific ABC transporter system in cells of Sphingomonas species A1. This is the first description of a bacterium with a pit on the cell surface and a pit-dependent endocytosic uptake system for macromolecules.

Crystallographic Studies of Mycobacterium tuberculosis Polyphosphate/ATP-NAD Kinase Complexed with NAD

NAD kinase from Mycobacterium tuberculosis (Ppnk) uses ATP or inorganic polyphosphate [poly(P)]. Ppnk overexpressed in Escherichia coli was purified and crystallized in the presence of NAD. Preliminary X-ray analysis of the resultant crystal indicate that the crystal belongs to hexagonal space group P6(2)22 and is holo-Ppnk complexed with NAD.